The present invention relates generally to the field of purinergic P2 receptors. More particularly, the present invention relates to novel purinergic P2X receptor compounds and production thereof. In further detail, the present invention relates to compounds, their intermediates, and enantiomers of both which have P2X receptor subtype 7 (P2X7) binding inhibition activity, and are useful for preventing and/or treating diseases associated with adenosine 5′-triphosphate or other natural or synthetic nucleotides.
P2 receptors have been generally categorized as either metabotropic nucleotide receptors or inotropic receptors for extracellular nucleotides. Metabotropic nucleotide receptors (usually designated P2Y or P2Yn, where “n” is a subscript integer indicating subtype) are believed to differ from inotropic receptors (usually designated P2X or P2Xn) in that they are based on a different fundamental means of transmembrane signal transduction: P2Y receptors operate through a G protein-coupled system, while P2X receptors are ligand-gated ion channels. The ligand for these P2X receptors is ATP, and/or other natural nucleotides, for example, ADP, UTP, UDP, and/or synthetic nucleotides, for example 2-methylthioATP.
A therapeutic role for P2 receptors has been suggested, for example, for cystic fibrosis (Boucher et al. (1995) in: Belardinelli et al. (eds) Adenosine and Adenine Nucleotides: From Molecular Biology to Integrative Physiology (Kluwer Acad., Norwell Mass.) pp 525–532), diabetes (Loubatieres-Mariani et al. (1995) in: Belardinelli et al. (eds), supra, pp 337–345), immune and inflammatory diseases (Di Virgilio et al. (1995) in: Belardinelli et al. (eds), supra, pp 329–335), cancer (Rapaport (1993) Drug Dev. Res. 28:428–431), constipation and diarrhea (Milner et al. (1994) in: Kamm et al. (eds.) Constipation and Related Disorders: Pathophysiology and Management in Adults and Children (Wrightson Biomedical, Bristol) pp 41–49), behavioral disorders such as epilepsy, depression and aging-associated degenerative diseases (Williams (1993) Drug. Dev. Res. 28:438–444), contraception and sterility (Foresta et al. (1992) J. Biol. Chem. 257:19443–19447), and wound healing (Wang et al. (1990) Biochim. Biophys. Res. Commun. 166:251–258).
At least seven P2X receptors, and the cDNA sequences encoding them, have been identified to date. P2X1 cDNA was cloned from the smooth muscle of the rat vas deferens (Valera et al (1994) Nature 371:516–519) and P2X.2 cDNA was cloned from PC12 cells (Brake et al. (1994) Nature 371:519–523). Five other P2X receptors have been found in cDNA libraries by virtue of their sequence similarity to P2X1 and P2X2 (P2X3: Lewis et al. (1995) Nature 377:432–435, Chen et al. (1995) Nature 377:428–431; P2X4: Buell et al. (1996) EMBO J. 15:55–62, Seguela et al. (1996) J. Neurosci. 16:448–455, Bo et al. (1995) FEBS Lett. 375:129–133, Soto et al. (1996) Proc. Natl. Acad. Sci. USA 93:3684–3688, Wang et al. (1996) Biochem. Biophys. Res. Commun. 220:196–202; P2X5: Collo et al. (1996) J. Neurosci. 16:2495–2507, Garcia-Guzman et al. (1996) FEBS Lett. 388:123–127; P2X6: Collo et al. (1996), supra, Soto et al. (1996) Biochem. Biophys. Res. Commun. 223:456–460; P2X7: Surprenant et al. (1996) Science 272:735–738). For a comparison of the amino acid sequences of rat P2X receptors see Buell et al. (1996) Eur. J. Neurosci. 8:2221–2228.
Native P2X receptors form rapidly activated, nonselective cationic channels that are activated by ATP. Rat P2X1 and rat P2X2 have equal permeability to Na+ and K+ but significantly less to Cs+. The channels formed by the P2X receptors generally have high Ca2+ permeability. The cloned rat P2X1, P2X2 and P2X4 receptors exhibit the same permeability for Ca2+ observed with native receptors. However, the mechanism by which P2X receptors form an ionic pore or bind ATP is not known.
A variety of tissues and cell types, including epithelial, immune, muscle and neuronal, express at least one form of P2X receptor. The widespread distribution of P2X4 receptors in the rat central nervous system suggests a role for P2X4-mediated events in the central nervous system. However, study of the role of individual P2X receptors is hampered by the lack of receptor subtype-specific agonists and antagonists. For example, one agonist useful for studying ATP-gated channels is α,β methylene ATP (meATP). However, the P2X receptors display differential sensitivity to the agonist with P2X, and P2X2 being meATP-sensitive and insensitive, respectively. Furthermore, binding of meATP to P2X receptors does not always result in channel opening. The predominant forms of P2X receptors in the rat brain, P2X4 and P2X6 receptors, cannot be blocked by suramin or PPADS. These two forms of the P2X receptor are also not activated by meATP and are, thus, intractable to study with currently available pharmacological tools.
The functional properties of the P2X1-6 receptors are fundamentally similar to those of the other two ionotropic receptors, the nicotinic and excitatory amino acid receptors, and are then relatively impermeable to cations that are more than about 200 D.
One of the most interesting members of the ionotropic P2X family is the P2X7 receptor. Di Virgilio, F., et. al. (1998) Cell Death and Differentiation 5:191; Di Virgilio, F., et. al. (1995) Immunology Today 16:524. The P2X7 ionic channel differs strikingly from these channels and it is formed by the aggregation of an unknown number of subunits each 595 aminoacids (AA) long (200 AA longer than the other six P2X receptor), and upon stimulation by high concentrations of extracellular ATP generates a nonselective membrane pore of variable size (3–5 nm) and permeable to hydrophilic molecules with molecular weight up to 900 Dalton. Suprenant, A., et. al. (1996) Science 272:735.
Selective activation of the P2X7 receptor in mycobacterial-infected cells may provide a new therapy for tuberculosis, as well as an effective anticancer agent for many tumors that are rich in P2X7 receptor. This receptor is mainly, if not exclusively, expressed by mononuclear phagocytes, where it mediates cytotoxic responses, cytokine release and cell fusion. Di Virgilio, F., et. al., (1998) Drug Dev. Res. 45:207; Falzoni, S., et. al. (1995) J. Clin. Invest. 95:1207. Activation of the P2X7 receptor in macrophages and microglial cells causes a large and rapid release of mature interleukin-1β in response to lipopolysaccaride (LPS) stimulation.
IL-1β is of prime importance in the induction of the immune responses, including facilitating response to antigens, synthesis of prostaglandins, proliferation of fibroblasts, blood neutrophils, and inducing the synthesis of other cytokines. Due to likely involvement in immunomodulation and in the inflammatory reaction, it would be of the most importance to develop selective P2X7 antagonists.
Human macrophages have proven very useful for the evaluation of P2X7 agonists and antagonists. Compound KN62 (1-N,0-(bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl)-4-phenylpiperazine) is one of the most potent antagonists for the P2X7 receptor with complete inhibition at the concentration of 500 nM. Gargett, C. E. et. al. (1997) B. J. Pharmacol. 120:1483. KN62 is a specific cell-permeable inhibitor of the autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMK II) and may be useful as a pharmacological tool for evaluating the role of CaMK II. Hidaka, H. et. al. (1992) Ann. Rev. Pharmacol. Toxicol., 32:377 (1992).
CaMK II is one of the important kinases whose response seems to be mediated by calmodulin, and that is proposed as a regulator for the synthesis and vescicular release of neurotransmitters. The same compound KN62 significantly inhibited both stimulated catecholamine release and secretory function via the direct blockade of activated Ca2+ influx. Maurer, J. A. et. al. (1996) J. Neurochem 66:105. KN62, at non-cytotoxic concentration (2 μM), enhanced etoposide (VP-16) cytotoxicity in Adriamycin-resistant cells (HL-60) and this is due to enhancement (from 2- to 4-fold) of VP-16 induced topoisomerase II-mediated DNA cleavable complex formation. Kawamura, K., et. al. (1996) Biochem Pharmacol. 12:1903. The DNA damage induced by VP-16 in the presence of KN62 resulted in the rapid introduction of apoptosis and depletion of cell in “S phase” of the cell cycle.
Thus, what is needed is specific agonists and antagonists for the P2X7 receptor subtype and, in particular, agents that will be effective in vivo, useful in the treatment of patients, as well as methods for identifying the P2X7 receptor-specific agonist and antagonist compounds.